Background:Fenugreek (Trigonella foenum-graecum L.) is an important medicinal plant that is a source of diosgenin, trigonelline and other valuable components. Fenugreek seeds is used for its pharmaceutical and nutraceutical properties.Objective: The aim of present study was to investigate the genetic diversity within and between different populations of Trigonella foenum-graecum using DNA markers and phytochemical characteristics. Methods: The total genomic DNA was isolated from 12 populations of Trigonella foenum-graecum collected from different regions in Iran. After optimizing PCR, 10 ISSR and 10 SCoT primers were used to amplify the genomic DNA of all genotypes. To investigate genetic distance, cluster analysis based on Jaccard similarity coefficients and UPGMA algorithm calculated for the 12 genotypes. Also, the trigonelline content was determined between 2 populations which had the highest genetic distance.Results: Analysis of banding patterns of 10 SCoT primers revealed 92 polymorphic bands with an average of 9.2 fragments per prime. A total of 79 bands were generated from 10 ISSR primers, out of which 55 (68.21%) were polymorphic bands. The average values of dissimilarities based on the SCoT and ISSR data were 0.60 and 0.44, respectively. Also, shoot trigonelline content was significantly different between genotypes with the highest genetic distance. Conclusion: Our results revealed a high level of genetic variation among fenugreek populations. The findings also indicated that SCoT technique is more informative than ISSR technique for evaluation of genetic diversity and relationships among fenugreek populations. The genotypes with the highest genetic distance based on SCoT data, were noticeably different in respect of trigonelline content.